WO1993022459B1

WO1993022459B1 - Oligonucleotides and nucleic acids for detection of ureaplasma urealyticum

Info

Publication number: WO1993022459B1
Application number: PCT/US1993/003923
Authority: WO
Filing date: 1993-04-26
Publication date: 1995-12-07
Anticipated expiration: 1994-10-27

Abstract

The present invention is directed to a rapid and sensitive method for detecting Ureaplasma urealyticum using U. urealyticum-specific probes and oligonucleotides. In particular a target sequence can be amplified by in vitro nucleic acid amplification techniques or directly detected by nucleic acid hybridization using the subject probes and oligonucleotides. U. urealyticum-specific nucleic acids which do not recognize or hybridize to genomic nucleic acid of Mycoplasma species are also provided.

Claims

AMENDED CLAIMS[received by the International Bureau on 10 December 1993 (10.12.93); original claims 1, 3, 6, 10, 14, 18, 20, 23, 24, 31-34, 37, 40 and 42 cancelled; rernaining claims amended and renumbered as claims 1-34 (6 pages)]

1. A method of detecting Ureaplas a urealyticum involving in vitro nucleic acid amplification which comprises: c (a) amplifying a U. urealyticum target nucleic acid by contacting a sample to be tested for the presence of U. urealyticum with at least one oligonucleotide for a time and under conditions sufficient to produce RNA or DNA copies of said target ₀ nucleic acid; wherein said oligonucleotide comprises a nucleotide sequence selected from genomic U. urealyticum DNA such that said oligonucleotide is sufficiently complementary to hybridize to said target nucleic acid to permit said amplification; and wherein said

■_jπ oligonucleotide does not hybridize to genomic DNA of M. genitalium, M. hominis, M. hyorhinis, M. orale, M. pneumoniae, and M. salivarium; and (b) detecting said copies.

2. A method of detecting Ureaplasma urealyticum 20 by nucleic acid hybridization which comprises:

(a) contacting a U. urealyticum target nucleic acid from a sample to be tested for the presence of U. urealyticum with a nucleic acid probe for a time and under conditions sufficient to permit hybridization _c between said nucleic acid probe and said target nucleic acid; wherein said nucleic acid probe comprises a nucleotide sequence selected from genomic U. urealyticum DNA such that said probe is sufficiently complementary to hybridize to said target nucleic acid and wherein

-- said probe does not hybridize to genomic DNA of M.

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qenitalium, M. hominis, M. hyorhinis, M. orale, M. pneumoniae, and M. salivarium; and

(b) detecting or measuring said hybridization.

3. The method of Claim 1 or 2 wherein said U. c urealyticum DNA comprises the U. urealyticum DNA inserted in plasmid pUP18, ρCU3900, pCU2800_f or pCU700.

4. The method of Claim 1 or 2 wherein said U. urealyticum DNA comprises the nucleotide sequence of SEQ ID N0:1 or a portion thereof. 0

5. The method of Claim 1 or 2 wherein said U. urealyticum DNA comprises the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

6. The method of Claim 1 or 2 wherein said target nucleic acid comprises the U. urealyticum DNA inserted in plasmic pUPlδ, pCU3900, pCU2800, or pCU700.

7. The method of Claim 1 or 2 wherein said target nucleic acid comprises the nucleotide sequence of SEQ ID NO:l or a portion thereof. o

8. The method of Claim 1 wherein said target nucleic acid comprises nucleotides 11 to 197 of SEQ ID NO:l.

9. The method of Claim 1 wherein said oligonucleotide is sufficiently complementary to

2c hybridize to the U. urealyticum DNA inserted in pUPlδ, PCU3900, pCU2800, or pCU700.

10. The method of Claim 1 wherein said oligonucleotide comprises a portion of the nucleotide sequences from SEQ ID NO:l.

--_Q 11- The method of Claim 1 wherein two oligonucleotides are used and one oligonucleotide

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2 comprises the nucleotide sequence of SEQ ID NO:2 and the other oligonucleotide comprises the nucleotide sequence Of SEQ ID NO:3.

12. The method of Claim 2 wherein said probe is 5 sufficiently complementary to hybridize to the U. urealyticum DNA inserted in pUP18, pCU3900, pCU2800,or PCU700.

13. The method of Claim 2 wherein said probe comprises a portion of the nucleotide sequences from SEQ 0 ID NO:l.

14. The method of Claim 2 wherein said probe comprises the nucleotide sequence of SEQ ID N0:l_f SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID

NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

15. The method of Claim 1 or 2 wherein said nucleotide sequence is sufficiently complementary when said nucleotide sequence is from about 70% to about 90% complementary to said U. urealyticum DNA.

16. The method of Claim 1 or 2 wherein said 0 sample is an animal body fluid, an animal secretion, an animal tissue, a culture medium or a transport medium.

17. The method of Claim 1 wherein said amplifying is accomplished by polymerase chain reaction, transcription-based amplification, self-sustained sequence replication, ligase-based amplification, Qβ- replicase RNA replication or transcription.

18. The method of Claim 1 wherein said amplifying enzymatic synthesis is performed by Escherichia coli DNA polymerase I, Klenow fragment of E.

■₃₀ ^co^- ^DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, Thermus aquaticus DNA polymerase, 1 Thermococcus litoralis DNA polymerase, SP6 RNA polymerase, T7 RNA polymerase, T3 RNA polymerase, T4 polynucleotide kinase, Avian Myeloblastosis Virus reverse transcriptase, Moloney Murine Leukemia Virus

5 reverse transcriptase, T4 DNA ligase, E. coli DNA ligase or Qβ replicase.

19. The method of Claim 2 wherein a reporter molecule is attached to said probe.

20. The method of Claim 19 wherein said reporter 0 molecule is a fluorophore, a bioluminescent molecule, a chemiluminescent, biotin, avidin, streptavidin, an enzyme, or a radioisotope.

21. The method of Claim 2 wherein said hybridization of solid-phase-based hybridization, solution hybridization, or in situ hybridization.

22. The method of Claim 1 wherein said oligonucleotide comprises an additional nucleotide sequence to facilitate said amplification.

23. The method of Claim 22 wherein said sequence o is ^an RNA polymerase binding site or a Qβ replicase binding site.

24. An isolated nucleic acid which comprises a sufficiently complementary nucleotide sequence selected from genomic U. urealyticum DNA such that said nucleic

2 acid can hybridize to said U. urealyticum DNA and cannot hybridize to genomic DNA of M. genitalium, M. hominis, M. hyorhinis, M. orale, M. pneumoniae, and M. salivarium.

25. The nucleic acid of Claim 24 wherein said U. -,_Q urealyticum DNA inserted in plasmic pUP18, pCU3900,

PCU2800, or pCU700.

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26. The nucleic acid of Claim 25 which is pUPlδ, PCU3900, pCU2800, or pCU700.

27. The nucleic acid of Claim 24 wherein said U. urealyticum DNA comprises the nucleotide sequence of SEQ

5 ID NO:1 or a portion thereof, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO: 9.

28. A compartmentalized kit for detection of U. urealyticum comprising a first container adapted to 0 contain at least one of the nucleic acids of Claim 24.

29. The kit of Claim 28 adapted to contain another container having U. urealyticum nucleic acid standard.

30. The kit of Claim 29 wherein said U. 5 urealyticum nucleic acid is U. urealyticum rRNA, U. urealyticum genomic DNA or an isolated DNA comprising SEQ ID NO:l.

31. The kit of Claim 28 having another container adapted to contain a reagent for in vitro nucleic acid o amplification.

32. The kit of Claim 31 wherein said reagent is an enzyme which is Escherichia coli DNA polymerase I, Klenow fragment of Escherichia coli DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, Thermus aquaticus DNA 5 polymerase, Thermococcus litoralis DNA polymerase, SP6 RNA polymerase, T7 RNA polymerase, T3 RNA polymerase, T4 polynucleotide kinase, Avian Myeloblastosis Virus reverse transcriptase, Moloney Murine Leukemia Virus reverse transcriptase, T4 DNA ligase, Escherichia coli _Q ^DNA ligase or Qβ replicase.

5 -

5 2

33. The kit of Claim 28 wherein said nucleic acid is linked to a reporter molecule.

34. The kit of Claim 33 wherein said reporter molecule is a fluorophore, a bioluminescent molecule, a chemiluminescent molecule, a radioisotope, biotin, avidin, streptavidin, or an enzyme.